Fibroblastos

Evidence that fibroblasts derive from epithelium during tissue fibrosis

Masayuki Iwano1, David Plieth1, Theodore M. Danoff2,3, Chengsen Xue1,Hirokazu Okada3,4 and Eric G. Neilson1,3

1Department of Medicine, and Department of Cell and Developmental Biology, Vanderbilt University, Nashville, Tennessee, USA

2 GlaxoSmithKline, Philadelphia, Pennsylvania, USA

3 Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA

4 Department of Nephrology, Saitama Medical College, Irumagun, Japan

Address correspondence to: Eric G. Neilson, Department of Medicine, D-3100 Medical Center North, Vanderbilt University Medical Center, Nashville, Tennessee 37232-2358, USA. Phone: (615) 322-3146; Fax: (615) 343-9391; E-mail: Eric.Neilson@Vanderbilt.edu.

Published August 1, 2002

Received for publication March 25, 2002, and accepted in revised form June 11, 2002.

Interstitial fibroblasts are principal effector cells of organ fibrosis in kidneys, lungs, and liver. While some view fibroblasts in adult tissues as nothing more than primitive mesenchymal cells surviving embryologic development, they differ from mesenchymal cells in their unique expression of fibroblast-specific protein-1 (FSP1). This difference raises questions about their origin. Using bone marrow chimeras and transgenic reporter mice, we show here that interstitial kidney fibroblasts derive from two sources. A small number of FSP1+, CD34– fibroblasts migrate to normal interstitial spaces from bone marrow. More surprisingly, however, FSP1+ fibroblasts also arise in large numbers by local epithelial-mesenchymal transition (EMT) during renal fibrogenesis. Both populations of fibroblasts express collagen type I and expand by cell division during tissue fibrosis. Our findings suggest that a substantial number of organ fibroblasts appear through a novel reversal in the direction of epithelial cell fate. As a general mechanism, this change in fate highlights the potential plasticity of differentiated cells in adult tissues under pathologic conditions.

See the related Commentary beginning on page 305.

Introduction

Cell fate pathways for epithelial tissues have overlapping complexities on many levels (1). Pathway integration ultimately determines the migration and interaction of progenitor cells under the control of genetic and morphogenic cues, the timed partitioning of cellular determinants, and plasticity among lineages until terminal differentiation shapes final structure and function (2, 3). With evolving tissue maturity, epithelial units organize as repeating structures, and fibroblasts come to reside in the interstitial spaces that form between functional units. Unfortunately, the order and assembly of these patterned events are not well understood (4, 5); for that matter, not all cells have been studied. The origin of interstitial fibroblasts, for example, has been largely overlooked, and their lineage is inconclusive (6). We undertook the present study because recent availability of new fibroblast markers has reduced the difficulty in addressing this issue (7, 8).

Two hypotheses emerge regarding the origin of adult fibroblasts. One hypothesis argues that marrow stromal cells (MSCs) are progenitors for tissue fibroblasts that then shuttle through the circulation to populate peripheral organs (6, 9–11). While MSCs can migrate to remote tissues and clearly develop a fibroblastic phenotype in culture (6), no evidence exists to show they engage in tissue fibrosis after migration. In fact, most of the recent interest in MSCs focuses on their capacity to give rise to more differentiated cells in nonhematogenous organs (5, 12, 13). A second hypothesis favors epithelial-mesenchymal transition (EMT) in the local formation of interstitial fibroblasts from organ epithelium (7). While many neoteric cell lineages migrate during embryogenesis to new locations using a fate pathway that involves EMT (14, 15), such transitions in mature tissues are less well appreciated. However, transitions do occur among adult cells (5), particularly during oncogenesis (16) and fibrotic tissue repair following injury — a process known as fibrogenesis (7, 17).

The appeal of an argument for EMT in the formation of fibroblasts is its simplicity; fibroblast dispersal in local interstitial spaces is assured by local epithelium, particularly when fibroblasts are needed for fibrogenesis. Indirect support for this notion stems from earlier work that identified fibroblast-specific protein-1 (FSP1) as an EMT marker in cultured epithelial cells undergoing transition to fibroblasts (18), as well as histologic evidence in vivo that epithelial units expressing FSP1 disaggregate as organ tissues dedifferentiate during the early stages of fibrogenesis (7, 19).

Epithelial cells sit on and attach to basement membranes that provide context and architectural stability for the cell-cell contact emblematic of this phenotype. When basement membrane is damaged by proteases or disrupted by alterations in assembly, epithelia begin to express cytokines that initiate EMT (20). Growth factors such as TGF-β, EGF, and FGF-2 facilitate EMT by binding epithelial receptors with ligand-inducible intrinsic kinase activity (16, 21, 22). The activation of Ras and Src pathways (16) and a shift in the balance of small GTPase activity (23) provide important transcriptional signals for loss of adhesion (24) and induction of EMT in cultured cells. In performing these functions, TGF-β and EGF also induce the expression of FSP1 in transitioning tubular epithelium (18). FSP1 is a fibroblast-specific protein in the S100 class of cytoplasmic, calcium-binding proteins (7). The members of this family have been implicated in microtubule dynamics, cytoskeletal membrane interactions, calcium signal transduction, p53-mediated cell cycle regulation, and cellular growth and differentiation. While the precise function of FSP1 and its homologues is not entirely clear, its interaction with non-muscle myosin II, tropomyosin, actin, or tubulin, and the inducibility of rearrangements in F-actin stress fibers suggest that FSP1 may be associated with mesenchymal cell shape and motility. Inhibition of EMT in cultured cells by blocking FSP1 expression implies a direct role in reshaping cytoskeletal architecture (18).

While EMT is well described in cultured cells, it remains to be shown whether such events occur in adult vertebrate tissues. Furthermore, although fibroblasts readily proliferate in culture when bombarded with cytokines and serum, they are generally quiescent in normal tissue. In preliminary in vivo experiments in normal mice expressing a transgene in tissue fibroblasts that encodes for thymidine kinase under the control of the FSP1 promoter (25), FSP1+ fibroblasts were not reduced in number by DNA chain termination until they randomly entered the cell cycle after more than 4–6 weeks of exposure to nucleoside analogues (data not shown). Therefore, in order to examine their in vivo derivation in a shorter time frame, we studied the appearance of new fibroblasts in a model of experimental renal fibrosis using specific protein markers. This approach, described below, also allowed us to evaluate the functional contribution of new fibroblasts identified from multiple sources.

Methods

Transgenic mice. Two sets of transgenic mice were constructed for these experiments. In the γGT.Cre mouse, the Cre transgene was removed from pMCCre (26) by digestion with MluI, and the overhang ends were blunted with Klenow fragment. A second vector, pγGT-GH, containing the rat γGTpromoter and a human growth hormone polyadenylation site (27), was digested at the XbaI site between the promoter and polyadenylation sequences, and the ends were also blunted with Klenow fragment. The blunted Cre transgene was then ligated to the blunted γGT promoter plasmid, and positive clones were selected for proper orientation. The chosen construct was then linearized using NotI and HindIII, and purified DNA was injected into B6 × SJL zygotes. The resulting progeny were crossed to Balb/c mice and selected by Southern blot and PCR (7, 8). The second transgene inserted into the FSP1.GFP mouse was assembled using pEGFP-N1 (Clontech, Palo Alto, California, USA) as a convenience vector. After deletion of the CMV promoter by digestion with AgeI and PstI and inserting a linker with XhoI and SfiI restriction sites, the vector was digested with EcoRI (5′) and BamHI (3′) in the region 5′ of the green fluorescent protein (GFP) gene. The EcoRI-to-NcoI FSP1 promoter fragment, with the NcoI site blunted by mung bean nuclease to remove the first ATG, was excised from a donor vector, FSP1.TK, (7, 8) using EcoRI (5′) and BamHI (3′) and then inserted into the GFP vector. Digestion of the final product with EcoRI and DraIII, (a site downstream of the polyadenylation signal), produced a linearized product for injection into B6D2 zygotes. Potential recombinant progeny were crossed to a Balb/c background and selected by Southern blot and PCR analysis of tail DNA. After second-generation mating, expression of GFP via the transgenic promoter is visible in the eyes of the mice under Woods illumination (see Figure 3b). Transgenic protocols were approved by the Instructional Animal Care and Use Committee (IACUC) at the University of Pennsylvania and Vanderbilt University.

Figure 3

Characterization of FSP1.GFP transgenic mice. (a) Plasmid map of the FSP1.GFP transgene injected into B6D2 zygotes for the production of transgenic mice. Ex 1, exon 1; In 1, intron 1. (b) Adult FSP1.GFP transgenic mice express GFP protein in all tissue fibroblasts. In adult FSP1.GFP mice, the eyes are demonstrably green under a Woods light compared with the wild-type because the GFP+ fibroblasts in the cornea cast a green tint. (c) RT-PCR

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